We are focusing our studies on proving or disproving the hypothesis that TRPC type cation channels are the mediators of store depletion activated calcium entry. In earlier studies we had discovered six of the seven TRPC channels, cloned full length cDNAs of four (TRPC1, TRPC2, TRPC3 and TRPC6) and shown them tobe activated by maneuvers that stimulate the Gq-PLCb-IP3 meditaed depletion of calcium stores. Focusing on TRPC3, and showed that peptides of the IP3 receptor that ionteract in vitro with TRPC3 segemnts (GST pull-down) affect store depletion activated calcium entry. But direct activation of TRPC3 upon thapsigargin stimulted store depletion indepndent of G protein-PLCbeta activation failed to show the classical capacitative calcium entry response. During this last year we discovered that TRPC3 is subject to phosphorylation on several tyrosines located on its N-terminus. Of these, tyrosine 226 is critical for function possibly involving phosphorylationby the c-src tyrosine kinase:1. TRPC3 expressed in cells lacking the src, yes and fyn tryosine kinases (SYF cells) is resistant to activation by Gq-PLCbeta stimualting maneuvers 2. expression in SYF cells trabnsfcted with src (SRC+, yes-, fyn ? cells) renders the channel responsive to agonist stimulation, 3. TRPC3 with a Tyr-226 to Phe mutation is unresponsive in wild type fibroblasts (src+, yes+, fyn+ cells) or HEK cells, and 4. an inactive TRPC3 channel in which all 11 N-terminal ttyrosines have been mutated to phenyalanine, becomes responsive to agonist stimulation upon mere back-mutation of Phe 226 to Tyr226. We also discovered the existence of an N-terminally extended form of TRPC3 (72 amino acids longer, one more tyrosine) in man, mouse and rat. We are currently studying the properties of this novel N-terminally extended TRPC3, especially whether it responds to thapsigargin-induced store depletion. We are also working on generating conditional knockout mice targeted for inducible inactivation of TRPC3, TRPC7 and TRPC5. These will add to our previously generated clasical knockouts of TRPC1 and TRPC6 and will serve to further investigate the physiological roles of the TRPC family of TRP channels.